1. First documentation of the existence of "Acquired Idiopathic Hypogamma-globulinemia" was provided by Prasad and Koza in 1954. (Prasad and Koza, Ann. Intern. Med., 41:629, 1954).
2. First description of the "Syndrome of Hypogammaglobulinemia, Splenomegaly and Hypersplenism" was published by Prasad, Reiner and Watson in 1957. (Prasad, Reiner and Watson, Blood, 12:626, 1957).
3. First description of "Polycythemia of Obesity" was documented by Weil and Prasad in 1955. (Weil and Prasad, Clin. Res., 3:194, 1955; Weil and Prasad, Ann. Intern. Med., 46:60, 1957).
4. Dr. Prasad described a method for determination of ultrafiltrable calcium and established partition of ultrafiltrable calcium and protein bound calcium in human plasma. Partition of serum calcium was unsettled until these studies were reported. Later, many other investigators confirmed their results. (Prasad and Flink, J. Appl. Physiol., 10:103, 1957; J. Lab. Clin. Med., 51:345,1958; and J. Lab Clin. Med., 52:1, 1958).
5. The studies of Dr. Prasad and his colleagues provided for the first time possible mechanisms accounting for post-hypercapnic ventricular fibrillation. Hyperkalemia and decrease in ultrafiltrable calcium (in spite of normal total calcium) in early post-hypercapnic phase were demonstrated. (Prasad, Brown and Flink, Am. J. Physiol., 190:459, 1957; Brown and Prasad, Am. J. Physiol., 100:462, 1957). These studies provided basis for proper management of post-hypercapnic syndrome in man.
6. First in vivo depolymerization of macroglobulins with penicillamine was reported by Bloch, Prasad and Anastasi in 1960. This observation has important therapeutic implications in macroglobulinemia inasmuch as many complications were due to presence of macroglobulins themselves. (Bloch, Prasad and Anastasi, J. Lab. Clin. Med., 56:212, 1960).
7. First description of "Human Zinc Deficiency Syndrome" was provided by Prasad, Halsted and Nadimi. (Prasad, Halsted and Nadimi, Am. J. Med., 31:532,1961; Prasad et al., J. Lab. Clin. Med., 61:537, 1963; Prasad et al., Arch. Int. Med., 111:407, 1963; Zinc Metabolism, C.C. Thomas, 1966). Growth retardation, a common phenomenon in developing countries, was related to dietary lack of zinc. Zinc supplementation reversed growth failure and hypogonadism (Sandstead HH, Prasad AS, Schulert AR, et al. Am J Clin Nutr 20:420-442, 1967. These observations have been confirmed by various other investigators and the existence of zinc deficiency in man has been recognized in many countries. It is estimated that nearly two billion subjects in developing countries may have deficiency of zinc.
8. Prasad, Oberleas and Halsted established method for assay of zinc in biological fluids by atomic absorption spectrophotometer when it first became available. (Prasad, Oberleas and Halsted, J. Lab. Clin. Med., 66:598,1965).
9. First definitive demonstration of enzyme changes in tissues due to zinc deficiency was documented by Prasad et al. (Prasad et al., J. Clin. Invest., 46:549, 1967). Several studies have been carried out in order to understand precise role of zinc in cell division and growth. Studies of Prasad et al. implicate enzymatic roles of zinc in nucleic acid metabolism (Prasad et al., J. Lab. Clin. Med., 77:144, 1977; Prasad et al., J. Lab. Clin. Med., 83:634, 1974). Later studies by Prasad et al. in rats indicate that thymidine kinase is a zinc dependent enzyme and that its activity is affected very early as a result of zinc deficient diet. This is the first demonstration of requirement of zinc for thymidine kinase, an enzyme regarded as essential for cell division and growth. (Prasad and Oberleas, J. Lab. Clin. Med., 83:634-639, 1974).
Although our studies showed that deoxythymidine kinase (TK) was a zinc dependent enzyme, the purified enzyme did not show presence of zinc in the molecule. We have now demonstrated that zinc is required for the gene expression of TK in HUT-78 cells (Th0 human malignant lymphoblastoid cell line). Decreased TK activity in zinc deficient cells in G1 phase affected adversely the DNA synthesis in S phase and delayed cell cycle. (Prasad et al, J. Lab. Clin. Med. 128:51-60,1996).
10. First evidence that zinc deficiency in rats produced behavioral changes was presented by Caldwell, Oberleas, Clancy and Prasad. (Caldwell, Oberleas, Clancy and Prasad, Proc. Soc. Exp. Biol. Med., 133:1417, 1970).
11. Mammen, Prasad, Barnhart and Au described Fibrinogen Detroit and it was documented that Arginine in 19th position was substituted by Serine in the chain of the fibrinogen molecule. This is the first demonstration that dysfibrinogenemia may be a `molecular disorder'. (Mammen, Prasad, Barnhart and Au, J. Clin. Invest., 48:235, 1969).
12. First evidence that sideroblastic gene in hereditary sideroblastic anemia, a sex-linked disorder, was linked to G6PD gene was presented by Prasad et al. (Prasad et al.,J. Clin. Invest., 47:1415, 1968).
13. The role of zinc in collagen synthesis has been established by Madrid, Prasad and Oberleas. Its essential role appears to be with fibroblastic proliferation related to its vital role in DNA synthesis. (Fernandez-Madrid, Prasad, and Oberleas, J. Lab. Clin. Med., 82:951, 1973).
14. Dr. Prasad and his colleagues have studied the effect of zinc on hemoglobin and changes in zinc metabolism in sickle cell disease. These studies were supported by National Heart and Lung Institute. Studies performed in collaboration with Dr. Brewer and his associates, show that in vitro zinc will shift the oxygen dissociation curve of the hemoglobin in the left. This property of zinc may be considered to be beneficial for patients with sickle cell disease inasmuch as their O2 dissociation curve is markedly right shifted and therefore the red cells deoxygenate readily in the tissue, thus accentuating sickling phenomena. (Oelshlegel, Brewer, Prasad, Knutsen, and Schoomaker, Biochem. Biophys. Res. Comm. 53:560, 1973; Oelshlegel, Brewer, Knutsen, Prasad, and Schoomaker, Arch. Biochem. Biophys., 163:742-748, 1974).
Delayed onset of puberty, short stature, hypogonadism and delayed healing of leg ulcers are common clinical findings in patients with sickle cell anemia (SCA). Similar clinical problems are also found in zinc deficient human subjects. Dr. Prasad has hypothesized that a secondary zinc deficient state may develop in SCA as a result of hyperzincuria secondary to hemolysis inasmuch as red blood cells are rich in zinc. His investigations showed that the plasma, RBC and hair zinc were decreased and the urinary zinc excretion increased in SCA patients in comparison to their controls.
A significant inverse correlation between RBC Zn and daily urinary Zn excretion in SCA suggested that hyperzincuria may have caused Zn deficiency in these patients. Carbonic anhydrase, a Zn metalloenzyme, correlated significantly with RBC Zn levels. RNase activity in the plasma was significantly greater in SCA subjects as compared to the controls. Zinc is known to be an inhibitor of the activity of this enzyme and an increased activity in Zn deficient tissues has been previously reported by Prasad, et al. Zinc therapy to zinc deficient SCA subjects has resulted in gain in height and weight and positive effect on growth of pubic, axillary, facial, body hair, dark adaptation, and cell mediated immunity. Beneficial effect on leg ulcers and apparent symptomatic improvement following Zn therapy has also been observed by Prasad et al., (Prasad et al., Clin. Chem., 21:582, 1975; Prasad et al., JAMA, 235:2396, 1976; Abbasi, Prasad, et al., Ann. Intern. Med., 85:601, 1976; Prasad et al., Am. J. Hem., 10:119, 1981; Warth, Prasad, et al., J. Lab. Clin. Med., 98:189, 1981; Prasad et al., Ann. Intern. Med., 100:367, 1984).
Brewer, G., Brewer, L. and Prasad, A.S., (J. Lab. Clin. Med., 90:549, 1977) have demonstrated that zinc affects the membrane of sickle cells beneficially and that administration of zinc orally suppresses significantly the number of irreversible sickle cells.
Prasad et al. have demonstrated that total protein, total collagen, RNA/DNA and the activity of deoxythymidine kinase, an enzyme required for DNA synthesis, are decreased in implanted sponge connective tissue in sickle cell anemia patients as compared to the controls (Prasad, Fernandez-Madrid, and Ryan, Am. J. Physiol., 236:E272, 1979). Inasmuch as deoxythymidine kinase is a zinc dependent enzyme, it is believed that zinc deficiency in sickle cell anemia patients may account for impaired collagen synthesis and a lack of prompt healing of leg ulcers.
Our studies in sickle cell disease patients have now shown that zinc supplementation (50 to 75mg daily orally), increased IL-2 production, decreased number of hospitalizations, decreased number of vasoocclusive pain crises, and decreased incidences of documented bacteriologically positive infections. (Prasad et al, Am. J. Hematol. 61:194-202,1999). Zinc supplementation also decreased oxidative stress and decreased generation of inflammatory cytokines in sickle cell disease patients. Bao B, Prasad AS, Beck FWJ, et al. Trans Res 52:67-80, 2008.
15. Detailed investigations of the effects of oral contraceptive agents on micro-nutrients have been conducted by Prasad and his colleagues. Changes in zinc, copper, folic acid and pyridoxine metabolism have been documented. These studies may have an important bearing on the determination of nutritional requirements of various vitamins and minerals in women using oral contraceptive agents. (Prasad et al., Am. J. Clin. Nutr., 28:377, 1975; Prasad et al., Am. J. Clin. Nutr., 28:385, 1975; Prasad et al., Am. J. Obstet. Gynecol., 125:1063, 1976; Lei, Prasad, Bowersox, and Oberleas, Am. J. Physiol., 231:98, 1976).
16. Dr. Prasad has successfully produced zinc deficiency experimentally in human volunteers by developing a semi-purified diet. The effects of a mild zinc deficient state in humans were studied. Four male volunteers received restricted zinc intake for several weeks under strict metabolic conditions. As a result of dietary zinc restrictions, a decrease in zinc concentration of plasma, erythrocytes, leukocytes, and urine was observed. Changes in the activities of zinc dependent enzymes in the plasma such as alkaline phosphatase and ribonuclease were also related to the dietary zinc status. An adverse effect of zinc restriction on total protein, total collagen, ribonucleic acid, and the activity of deoxythymidine kinase (a zinc dependent enzyme) in the sponge connective tissue of the two volunteers in whom this test was done was noted.
During the zinc restriction period, the ammonia level in the plasma was elevated. Weight loss occurred in all subjects as a result of dietary zinc restriction. Inasmuch as the zinc deficient state was mild, this study provides a basis for developing diagnostic criteria for zinc deficiency in humans. This study is still in progress and provides a unique method for studying the effects of marginal zinc deficiency in man directly and may provide basis for determination of zinc requirement in man. (Prasad et al., Ann. Intern. Med., 89:483, 1978). Our studies in human volunteers have also demonstrated that dietary zinc restriction can decrease sperm count and that oligospermia induced by a mild deficiency of zinc is a reversible process. Oligospermia thus seems to be a sensitive indicator of zinc deficiency (Abbasi, Prasad, and Rabbani, Trans. Assoc. Am. Phys., 92:292, 1979; Abbasi, Prasad, et al., J. Lab. Clin. Med., 96:544, 1980).
17. The occurrence of zinc deficiency in uremics has been documented. (Mahajan, Prasad et al., J. Lab.Clin. Med., 94:693, 1979; Briggs, Prasad et al., Kidney International, 21:827, 1982). These studies demonstrate that certain clinical features such as hypogonadism, poor appetite, taste abnormalities and hyperammonemia may be related to a deficiency of zinc. A double blind trial with zinc supplementation in chronic uremic patients has shown that taste abnormalities and testicular functions improve after zinc supplementation. (Mahajan, Prasad et al., Am. J. Clin. Nutr., 33:1517, 1980; Mahajan, Abbasi, Prasad et al., Ann. Intern. Med. 97:357, 1982; Mahajan, Prasad et al., Am. J. Clin. Nutr. 36:1177, 1982).
18. Studies by Prasad and his colleagues indicate that nucleoside phosphorylase may be a zinc dependent enzyme and that its activity may be adversely affected in zinc deficient animals and humans. This observation may provide possible explanation for T-Cell dysfunctions, which have been observed in zinc deficiency. (Prasad, and Rabbani, Trans. Assoc. Am. Phys., 94th session, p 314, 1981; Ballester and Prasad, Ann. Intern. Med. 98:180, 1983).
19. Hyperammonemia occurring as a result of zinc deficiency in man was first reported by Prasad and his colleagues (Prasad et al., Ann. Intern. Med., 899:483, 1978). This was soon confirmed in experimental animals (Rabbani and Prasad, Am. J. Physiol., 235:E203, 1978) and in zinc deficient sickle cell anemia patients (Prasad et al., Am. J. Hematol., 7:323, 1979) and chronic uremics (Mahajan, Prasad, et al., Am. J. Clin. Nutr., 36:1177, 1982).
Zinc produced a negative copper balance in patients with sickle cell disease, suggesting that it may be a useful agent to decrease copper burden in Wilson’s disease (Prasad, A.S., Brewer, G.J., Schoomaker, E.B., Rabbani, P. JAMA, 240:2166-2168, 1978).
20. Brewer, Hill, Prasad, Cossack and Rabbani (Ann. Int. Med., 99:314,1983) reported the beneficial use of zinc supplementation for the therapy of Wilson's disease. Brewer, et al succeeded in establishing zinc as RDA approved therapeutic modality for Wilson’s disease.
21. Studies carried out in collaboration with Dr. Dardenne and J. Bach showed that thymulin, a zinc dependent thymic hormone is a very sensitive indicator of mild human zinc deficiency state. In experimental human model, a mild state of zinc deficiency was induced by dietary means and this resulted in a marked decrease in the activity of serum thymulin. This was accompanied by an alteration of T helper to T suppressor ratio, an increase in T101-, sIg- cells, and decreased activity of IL-2 and NK cells. All these immunological parameters were reversed by zinc supplementation. These studies provide for the first time very important insight into the role of zinc in cell mediated immunity. These studies were presented at the Association of American Physicians meeting held at San Diego in 1987 and the manuscript was published in 1988 (Prasad et al, J.Clin.Invest. 82:1202-1210, 1988).
22. New techniques for the assay of zinc in lymphocytes, granulocytes, and platelets have been established in Dr. Prasad's laboratory. The cellular zinc levels are being utilized to diagnose mild and marginal deficiency of zinc in humans. This is an important advance, inasmuch as plasma zinc is not a sensitive indicator of zinc deficiency in humans (Wang H., Prasad AS, DuMouchelle E. J. Micronutrients Anal 5:181-190, 1989).
23. With the use of cellular zinc levels, six apparently normal human subjects were identified to have a mild deficiency of zinc. Nucleotide assay of lymphocytes in these subjects revealed that ATP/ADP ratio was decreased suggesting a lower energy level in the lymphocytes, and a significant accumulation of GTP and dGTP were observed in comparison to zinc sufficient controls. Nucleoside phosphorylase activity was decreased in zinc deficient lymphocytes, which may have caused an accumulation of toxic nucleotides in lymphocytes. These studies provide yet another mechanism by which a deficiency of zinc affects lymphocytes adversely. These studies were presented at the Central Society of Clinical Research meeting held in Chicago in November, 1987. Dr. Meftah, a resident in the Department of Medicine, presented this paper. Dr. Meftah also received 1987 Eli Lilly Award of the Central Society for Clinical Research for this work. (Meftah and Prasad, J. Lab. Clin. Med., 114(2):114-119, 1989).
24. We have studied extensively the role of zinc on immune functions in humans. In experimental model of human zinc deficiency, we have shown that in the peripheral blood mononuclear cells of zinc deficient cells the production of IL-2 and INF-γ are decreased but the production of IL-4, IL-6, and IL-10 are not affected, suggesting that there is a shift of cytokine production from Th1 to Th2 cells. NK cell lytic activity and pre-cytolytic T-cells are decreased in zinc deficiency. Thus besides a decrease in serum thymulin activity, the major effect of zinc deficiency is on Th1 cells which are affected adversely. The production of IL-1β from monocytes-macrophages are increased in zinc deficient subjects, suggesting that zinc deficiency leads to activation of monocytes-macrophages. These studies provide mechanisms of zinc action on cell mediated immunity in humans. (Prasad, J. Inf. Dis. 182 (suppl.):62-68, 2000, Beck FWJ, Prasad AS et al, Am. J. Physiol. 272:E1002-7, 1997, Prasad AS et al, Proc. Assoc. Am. Phys., 109:68-77, 1997).
25. Zinc and Immunity
When I was in the Middle East, I was struck by the observation that I never saw any zinc deficient dwarf who was older than 25 years of age. On extensive enquiries, I found out that they were dying because o bacterial, viral or parasitic infections. This led me to do extensive studies of immune functions as related to zinc in humans. We showed that I cell functions were affected adversely even when the deficiency of zinc was mild in humans. (Prasad et al, J Clin Invest, 1988). Characteristically during zinc deficiency in humans, the serum thymulin activity (a thymic hormone) was decreased which was restored following zinc supplementation. Our studies also showed that zinc deficiency caused an imbalance between TH1 and TH2 functions. The production of IFN-γ and IL-2 (products of TH1 cells) were decreased, whereas the production of IL-4, IL-6 and IL-10 (products of TH2) were not affected during zinc deficiency. T cell subpopulation studies revealed that the CD4 + CD45RA to CD4 + CD45RO ratio was decreased as a result of zinc deficiency, suggesting that zinc may be required for regeneration of new CD4 + T cells (CD4 + CD45RA). WE further documented that zinc deficiency decreased NK cell lytic activity and caused a decrease in the percentage of CD8 + CD73 + T cells which are known to be predominately precursors of cytotoxic T cells.
Recent studies in India, Bangladesh and other developing countries have shown that zinc supplementation decreases significantly morbidity and mortality in children with acute diarrhea and respiratory tract infections. Microsoft, Inc. founder Bill Gates, during his recent travels to Bangladesh and India became aware of zinc deficiency affecting millions of children in the developing countries and he has directed Melinda and Bill Gates Foundation to focus their attention on this problem globally. I have been appointed as their consultant to provide the foundation a landscape of zinc effect and how this can be used to decrease the morbidity and mortality in children.
Molecular mechanism of zinc in activation of NF-κB and IL-2 gene expression
26. Nearly 2000 transcription factors require zinc for their structural integrity; however, it is not known whether cellular zinc deficiency results in any change in activation of any of the transcription factors. Inasmuch as NF-κB binds to the promoter enhancer area of IL-2 and IL-2R genes, we investigated the effect of zinc deficiency on activation of NF-κB and its binding to DNA in HUT-78, a TH0 malignant human lymphoblastoid cell line. We showed for the first time that in zinc-deficient HUT-78 cells, phosphorylated IκB, and Ikk, ubiquitinated IκB and binding of NF-κB to DNA were all significantly decreased. Zinc increased the translocation of NF-κB from cytosol to nucleus. We also demonstrated that the binding of recombinant NF-κB (p50)2 to DNA in HUT-78 cells was zinc specific. We concluded that zinc plays an important role in the activation of NF-κB in HUT-78 cells (Prasad et al, J Blab Clin Med 2001;138:250-6). Production of IL-2 is decreased in zinc-deficient humans, and zinc is essential for IL-2 mediated T-cell activation. We used HUT- 78 to study the effect of zinc on IL-2 production in PHA/PMA activated T-cells. In zinc-deficient cells, the gene expression of IL-2 was decreased by 50% compared with that in zinc-sufficient cells. The effect of zinc was specific and it was at the transcriptional level. We also showed a significant effect of zinc on the gene expression of IL-2 receptors. Our studies demonstrated for the first time the role of zinc in gene expression of IL-2 and its receptors in HUT-78 cells. We also documented that the binding of NF-κB to DNA was adversely affected, thereby decreasing the gene expression of IL-2 and IL2R in zinc-deficient HUT-78 cells (Prasad et al, J Lab Clin Med 2002; 140;272-89.
27. Chronic generation of inflammatory cytokines and reactive oxygen species are implicated in atherosclerosis, aging, cancers, and other chronic diseases. We hypothesized that zinc induces A20 in premonocytic, endothelial, and cancer cells, and A20 binds to tumor necrosis factor (TNF)-receptor associated factor, and inhibits Ik kinase-a (Ikk-α)/nuclear factor-κB (NF-κB), resulting in downregulation of TNF-α and interleukin-1β (IL-1β). To test this hypothesis, we used HL-60, human umbilical vein endothelial cells, and SW480 cell lines under zinc-deficient and zinc-sufficient conditions in this study.
We measured oxidative stress markers, inflammatory cytokines, A20 protein and mRNA, A20–FRAF-1 complex, and Ikk-α/NF-κB signaling in stimulated zinc-deficient and zinc sufficient cells. We also conducted antisense A20 and siRNA studies to investigate the regulatory role of zinc in TNF-α and IL-1β via A20. We found that zinc increased A20 and A20–tumor necrosis factor-receptor associated factor-1 complex, decreased the Ikk-α/NF-κB signaling pathway, oxidative stress markers, and inflammatory cytokines in these cells compared with zinc-deficient cells. We confirmed that zinc induced A20 contributes to down-regulation of TNF-α and IL-1β by antisense and short interfering RNA A20 studies.
Our studies suggest that zinc suppresses generation of NF-κB–regulated inflammatory +cytokines by induction of A20.
28. Zinc deficiency impairs cellular immunity. Up-regulation of mRNA levels of IFN-γ, IL-12R2, and T-bet are essential for Th1 differentiation. We hypothesized that zinc increases Th1 differentiation via up-regulation of IFN-γ and T-bet expression. To test this hypothesis, we used zinc-deficient and zinc-sufficient HUT-78 cells (a Th0 cell line) under different condition of stimulation in this study. We also used TPEN, a zinc-specific chelator, to decrease the bioavailability of zinc in the cells. We measured intracellular free zinc, cytokines, and the mRNAs of T-bet, IFN-γ, and IL-12R2. In this study, we show that in zinc-sufficient HUT-78 cells (a Th0 cell line), mRNA levels of IFN-γ, IL-12R2, and T-bet in PMA/PHA-stimulated cells were increased in comparison to zinc-deficient cells. Although intracellular free zinc was increased slightly in PMA/PHA-stimulated cells, Con-A-timulated cells in 5 μM zinc medium showed a greater sustained increase in intracellular free zinc in comparison to cells incubated in 1 μM zinc. The cells pre-incubated with TPEN showed decreased mRNA levels of IFN-γ and T-bet mRNAs in comparison to cells without TPEN incubation.
We conclude that stimulation of cells by Con-A via TCR, release intracellular free zinc which functions as a signal molecule for generation of IFN-γ and T-bet, and IL-12Rβ2 mRNAs required for Th1 cell differentiation. These results suggest that zinc increase Th1 cell differentiation by up-regulation of IFN-γ and T-bet, and IL-12Rβ2 mRNAs.